Method for identification of anti-HIV human miRNA mimics and miRNA inhibitors and anti-HIV pharmaceutical compounds

ABSTRACT

The present invention relates to methods for the identification of anti-HIV miRNAs and anti-HIV pharmaceutical compounds using high-throughput screening methods, comprising: transfecting reporter cells with a panel of miRNAs, infecting the reporter cells with HIV, screening the cells to identify miRNAs that modulate HIV infection and identifying the specific pathways, nucleic acids and/or polypeptides that are targeted by the miRNAs. The invention further provides for the identification and screening of anti-HIV pharmaceutical compounds having known activity against the specific pathways, nucleic acids and/or polypeptides that are targeted by the miRNAs for efficacy in the treatment of HIV. The invention also provides for the use of miRNA mimics, miRNA inhibitors and pharmaceutical compounds (including oncology drugs and kinase inhibitors) in the treatment and/or prevention of HIV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/105,029, filed on Jun. 16, 2016, which is the U.S. national phaseapplication filed under 35 U.S.C. § 371 claiming benefit toInternational Patent Application No. PCT/IB2014/067019, filed on Dec.17, 2014, which is entitled to priority under 35 U.S.C. § 119(e) to ZAprovisional application no. 2013/09501, filed Dec. 17, 2013, thedisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

The present invention relates to methods for the identification ofanti-HIV miRNAs and anti-HIV pharmaceutical compounds usinghigh-throughput screening methods, comprising: transfecting reportercells with a panel of miRNAs, infecting the reporter cells with HIV,screening the cells to identify miRNAs that modulate HIV infection andidentifying the specific pathways, nucleic acids and/or polypeptidesthat are targeted by the miRNAs. The invention further provides for theidentification and screening of anti-HIV pharmaceutical compounds havingknown activity against the specific pathways, nucleic acids and/orpolypeptides that are targeted by the miRNAs for efficacy in thetreatment of HIV. The invention also provides for the use of miRNAmimics, miRNA inhibitors and pharmaceutical compounds in the treatmentand/or prevention of HIV infection.

Humans display a remarkably diverse susceptibility to infection, thefoundation of which lies in our genetic variation and ability to respondto selective pressures applied by various infectious agents. Theevolution of our complex and multi-player immune system underlines thedominance of the human host following a microbial infection. However,given the nature of obligate intracellular pathogens, their completereliance on host gene expression machinery has led to the evolution ofcomplex interplays between the two, such that pathogens actively andstrategically manoeuvre their way through the host terrain. Ourtraditional view of this terrain as being comprised of protein-codinggenes, translation intermediates (mRNAs) and protein counterparts is fartoo simplistic, particularly in the context of infection. The discoveryof the RNA interference (RNAi) pathway, for which the 2006 Nobel Prizewas awarded, has greatly enhanced our understanding of the host terrain.Small noncoding RNAs (ncRNAs) termed microRNAs (miRNAs) were shown to bekey regulators of gene expression that function within the RNAi pathwayto post-transcriptionally modulate mRNA stability and subsequenttranslation (Fire et al., (1998)). Indeed, it is now understood thatmiRNAs are able to rapidly, and with exquisite specificity, modulategene expression in response to numerous environmental cues in a highlycoordinated, complex and tissue-specific manner. Given the reliance ofintracellular pathogens on host gene expression machinery, the RNAipathway, and specifically miRNAs, are now understood to lie at the nexusof the host-pathogen interplay.

Only 2% of the metazoan genome encodes protein, yet more than 50% istranscribed and we have little knowledge regarding these transcriptsthat function in the absence of protein production. In fact, stablencRNA transcripts have been referred to as ‘dark matter’ within thecellular environment (Yamada et al., (2003)). Despite improvements inthe human draft genome sequence, ncRNAs remain difficult to define andthus quantify (Ponting et al., (2010)). However, numerous evolutionarystudies have revealed that ncRNAs are estimated to be expressed at4-fold excess compared to their protein-coding counterparts and arehighly conserved across eukaryotic genomes (Ponting et al., (2010)).Currently, ncRNAs have been classified by size into long ncRNAs(IncRNAs; >200 bp) or small ncRNAs (<200 bp) and comprise 26 or morefunctional categories reflecting something of the diversity of ncRNAfunction (Ponting et al., (2010)). In the case of humans, IncRNAscomprise about 48% of the genome (Lander et al., (2001)) while smallncRNAs including miRNAs, constitute a small but significant portion ofthe genome. While new ncRNAs are rapidly being uncovered, functionaldata remains sparse particularly at the host-pathogen interface.Endogenous miRNAs are usually transcribed from RNA polymerase IIpromoters (Lee et al., (2004)) as primary miRNA transcripts (pri-miRNAs)several kb in length, containing various stem-loop structures and ssRNAflanking segments. Pri-miRNAs are processed in two compartmentalizedsteps via the actions of distinct protein complexes (Lee et al., (2002))to form mature miRNAs that regulate post-transcriptional proteinsynthesis by base pairing to cognate mRNAs. Depending on the degree ofcomplementarity between the mature miRNA and its target, multiple mRNAsilencing modes can occur (Azuma-Mukai et al., (2008)). The initialbases from positions 2 to 7 of the mature miRNA are termed the ‘seed’sequence and they provide most of the pairing specificity. In somecases, complete pairing between the seed region and target mRNA issufficient to mediate cleavage of the cognate mRNA (Liu et al., (2004);Yekta (2004)). More typically for mammalian and viral mRNA targetshowever, cleavage activity is severely impaired by mismatched pairing inthe seed and other regions and translational inhibition occurs (Martinezand Tuschl (2004)). Intriguingly, since the complementary length of seedsequence required for miRNAs to target cognate mRNAs is short, eachmiRNA can target and modulate hundreds of transcripts. Indeed, currentestimates predict that thousands of human transcripts are regulated bymiRNAs (Farh et al., (2005); Lim et al., (2005)). Furthermore, a singlemiRNA can regulate multiple mRNA molecules that can in turn also beacted upon by numerous distinct miRNAs (Barbato et al., (2009)).Importantly, most miRNAs decrease target protein levels by less than2-fold (Baek et al., (2008)), but this non-linear tuning mechanism canstill exert a large physiological effect (Ebert and Sharp (2012)). Thus,the endogenous miRNA pathway represents a highly efficient system tosimultaneously fine-tune the expression of numerous genes as well asmodulate specific functional pathways.

Considering human cells encode >1000 miRNA species, many of whichfunction in innate immunity, it is unsurprising that pathogens (andviruses in particular) have evolved mechanisms to subvert these cellularcomponents (Cullen (2013)). The particular mechanisms by which virusesmanipulate the host immune system are as varied as the virusesthemselves but if one focuses on viral interactions with cellularmicroRNA machinery, the options are surprisingly minimized andconstrained to a fairly limited number of human viruses. Furthermore, asmiRNAs are expressed in a very tissue specific manner and can varydepending on the cell cycle stage, the interactions between host miRNAsand pathogens is clearly complex. The ability of a host organism tomount an innate immune response after pathogen infection is critical forsurvival and many cellular mRNAs that control host defences areregulated by miRNAs. The promiscuity of miRNAs in regulating their mRNAtargets coupled with their importance in posttranscriptional regulationof host gene expression make unraveling the role of miRNAs at thehost-pathogen interface extremely challenging. Resolving theseinteractions requires identification of the specific pathogen-encodedstimuli that induce changes in the host miRNome following infection,assessment of which transcripts are targeted by miRNAs as well as whichmiRNAs are responsible, quantification of the miRNA-induced changes tothe infection transcriptome, analysis of downstream effects on relatedprotein outputs, and validation of each step to ensure a robustunderstanding of such a complex network of interactions.

To identify host miRNAs required by the Human Immunodeficiency Virus(HIV-1) during infection, we conducted several genome-wide miRNA-basedscreens. We identified numerous host miRNAs that inhibit HIV-1replication or enhance activation of the HIV long terminal repeat (LTR)promoter either when the specific miRNA is over-expressed (thus boostingexpression levels of its endogenous miRNA sequence) or suppressed (thus‘sponging out’ or quenching its endogenous miRNA sequence).

Based on extensive literature searches, the human mRNA targets of eachof the HIV inhibitory miRNAs were identified. Intriguingly, many ofthese mRNA targets code for host proteins that play a role in thecellular response to DNA damage, repair and apoptosis. Given that thesecellular pathways are central to the development and control of manycancers, we conducted additional validation experiments which havehighlighted the close relationship between early HIV infection andabnormal cell survival phenotypes which are related to the DNA damagepathway and which are characteristic of many cancers. We thus sought toutilise compound libraries comprised of oncogenic and kinase-specificinhibitors, and tested for their ability to inhibit HIV infection.Several of the cancer-specific therapeutics inhibited HIV replication attargets and pathways analogous to those identified in the miRNA screens.Taken together our results have revealed novel host miRNAs as well asoncogenic compounds that can be repurposed for use in anti-retroviraltherapies targeted against HIV-1.

SUMMARY OF THE INVENTION

The present invention relates to a method for the identification ofanti-HIV miRNAs and anti-HIV pharmaceutical compositions.

According to a first aspect of this invention there is provided for amethod for the identification of anti-HIV miRNAs and anti-HIVpharmaceutical compounds, wherein the method comprises the steps of: a)providing a first batch of reporter cells and providing a panel ofmiRNAs, wherein the reporter cells are divided into a plurality ofsamples and wherein each sample is transfected with an miRNA from thepanel, b) infecting the transfected cells with HIV, c) screening thesamples to identify one or more (anti-HIV) miRNAs which modulate HIVinfection from the panel of miRNAs, and d) identifying a specificcellular pathway, a polynucleotide and/or a polypeptide which istargeted by an anti-HIV miRNA from step c.

In one embodiment of the invention the method further comprises thesteps of e) providing a second batch of reporter cells and providing apanel of pharmaceutical compounds with known pharmaceutical activityagainst the cellular pathways, the polynucleotides and/or thepolypeptides of step d), wherein the second batch of reporter cells aredivided into a plurality of samples and wherein each sample is treatedwith a pharmaceutical compound, f) infecting the second batch ofreporter cells with HIV, wherein the step of infecting the second batchof reporter cells may occur before or after the treatment with thepharmaceutical compound; and g) identifying a pharmaceutical compoundwhich has pharmaceutical activity against the specific cellular pathway,polynucleotide and/or polypeptide targeted by anti-HIV miRNA.

In one embodiment of the invention the miRNAs may be selected from miRNAmimics or miRNA inhibitors. Preferably, the miRNAs are human miRNAs.

In another embodiment of the invention an anti-HIV miRNAs whichmodulates HIV infection is selected from the group consisting of: i)anti-HIV miRNAs which are able to inhibit HIV entry into a cell, ii)anti-HIV miRNA molecules which are able to suppress activation of theHIV Long Terminal Repeat (LTR) promoter, iii) anti-HIV miRNA moleculeswhich are able to sensitise uninfected cells to apoptosis in response toinfection by HIV, and/or iv) anti-HIV miRNA molecules which are able topromote activation of the HIV LTR promoter.

In a further embodiment the anti-HIV miRNAs are selected from the groupconsisting of hsa-let-7a-5p, hsa-let-7d-5p, hsa-miR-23a, hsa-mir-29c*,hsa-miR-34c-3p, hsa-miR-92a-1-5p, hsa-mir-124a-3p, hsa-miR-124a,hsa-miR-125b-5p, hsa-miR-138, hsa-miR-146a, hsa-miR-149-3p, hsa-miR-150,hsa-miR-155. hsa-miR-193b-5p, hsa-miR-200c, hsa-miR-342-5p,hsa-miR-361-5p, hsa-miR-421, hsa-miR-423-3p, hsa-miR-504,hsa-miR-509-3p, hsa-miR-637, hsa-mir-650, hsa-miR-520d-5p, hsa-miR-1200,hsa-miR-1908, hsa-miR-1910, hsa-miR-2110, hsa-miR-3162, hsa-miR-3185,hsa-miR-3189, hsa-miR-3191, hsa-miR-3191-3p, hsa-miR-4259, andhsa-miR-4314.

In yet another embodiment of the invention the pharmaceutical compoundof step g) may be selected from the group consisting of: i)pharmaceutical compounds which are selectively toxic to cells infectedwith HIV, ii) pharmaceutical compounds which are able to sensitizeuninfected cells to apoptosis in response to infection by HIV, iii)pharmaceutical compounds which are able to suppress activation of theHIV LTR promoter in cells infected with HIV, iv) pharmaceuticalcompounds which are able to inhibit HIV entry into a cell, and/or v)pharmaceutical compounds which are able to prime uninfected cells tosuppress HIV LTR promoter activity in response to infection by HIV.

It will be appreciated that the pharmaceutical compound of step g) mayselected from the group consisting of an oncology drug or a kinaseinhibitor. The oncology drug may be selected from the group ofpharmaceutical compounds consisting of abiraterone, aminolevulinic acidhydrochloride, cisplatin, dactinomycin, dexrazoxane, erlotinibhydrochloride, everolimus, floxuridine, gefitinib, ifosfamide,plicamycin, temozolomide, thalidomide, vemurafenib, vincristine sulfate,vorinostat, and pharmaceutically acceptable salts thereof. In a furtherembodiment the kinase inhibitor may be selected from the groupconsisting of A-674563, AT7519, SNS-032, aurora A inhibitor I,crenolanib, foretinib, GSK2126458, NVP-BHG712, LDN193189, PIK-75,ponatinib, saracatinib, vargatef, and WZ4002.

According to a second aspect of the invention there is provided for anmiRNA mimic or an miRNA inhibitor selected from the group consisting ofhsa-let-7a-5p, hsa-let-7d-5p, hsa-miR-23a, hsa-miR-92a-1-5p,hsa-mir-124a-3p, hsa-miR-124a, hsa-miR-138, hsa-miR-146a,hsa-miR-149-3p, hsa-miR-150, hsa-miR-193b-5p, hsa-miR-342-5p,hsa-miR-421, hsa-miR-423-3p, hsa-miR-509-3p, hsa-miR-637, hsa-ir-650,hsa-miR-520d-5p, hsa-miR-1908, hsa-miR-2110, hsa-miR-3162, hsa-miR-3185,hsa-miR-3189, hsa-miR-3191, hsa-miR-3191-3p, hsa-miR-4259, andhsa-miR-4314 for use in the suppression of HIV replication in a cell. Athird aspect of the invention provides for an miRNA mimic or an miRNAinhibitor selected from the group consisting of hsa-mir-29c*,hsa-miR-193b-5p, hsa-miR-421, hsa-miR-1908, and hsa-miR-3189 for use ininducing apoptosis in a cell in response to HIV infection of the cell.Yet a further aspect of the invention provides for an miRNA mimic or anmiRNA inhibitor selected from the group consisting of hsa-miR-34c-3p,hsa-miR-125b-5p, hsa-miR-150, hsa-miR-155, hsa-miR-200c, hsa-miR-361-5p,hsa-miR-504, hsa-miR-1200 and hsa-miR-1910 for use in increasing thelevel of HIV replication in a cell.

In another aspect of the invention there is provided for a compound or apharmaceutically effective salt thereof selected from the groupconsisting of foretinib, GSK2126458, NVP-BHG712, ponatinib, saracatinib,temozolomide, thalidomide, vargatef, vincristine sulfate, and WZ 4002for use in the prevention of HIV infection in a subject. In yet anotheraspect of the invention the compound or pharmaceutically acceptable saltthereof, is selected from the group consisting of A-674563,dactinomycin, ifosfamide, cisplatin, dexrazoxane, everolimus andvemurafenib for use in the suppression of HIV infection in an HIVinfected subject. A further aspect of the invention provides for acompound or a pharmaceutically acceptable salt thereof, selected fromthe group consisting of A-674563, abiraterone, aminolevulinic acidhydrochloride, AT7519, aurora A inhibitor I, crenolanib, erlotinibhydrochloride, floxuridine, gefitinib, LDN193189, PIK-75, plicamycin,SNS-032, and vorinostat for use in inducing apoptosis in a cell inresponse to HIV infection of the cell in a subject.

BRIEF DESCRIPTION OF THE FIGURE

Non-limiting embodiments of the invention will now be described by wayof example only and with reference to the following FIGURE:

FIG. 1: Agonist-Antagonist approach using miRNA mimic and inhibitormolecules

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown.

The invention as described should not be limited to the specificembodiments disclosed and modifications and other embodiments areintended to be included within the scope of the invention. Althoughspecific terms are employed herein, they are used in a generic anddescriptive sense only and not for purposes of limitation.

As used throughout this specification and in the claims which follow,the singular forms “a”, “an” and “the” include the plural form, unlessthe context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of the terms“comprising”, “containing”, “having” and “including” and variationsthereof used herein, are meant to encompass the items listed thereafterand equivalents thereof as well as additional items.

Provided herein is a high-throughput screening method for screening alibrary of miRNA mimics and inhibitors in 96 well plates in duplicate inan engineered reporter cell line. This engineered reporter cell linestably expresses an HIV LTR promoter upstream of hGFP. Reporter cells inexperimental wells were transfected with a specific miRNA mimic orinhibitor 48 hours prior to infection with HIV. Cells were then infectedwith HIV for 48 hours and samples processed for imaging. Images wereacquired using an automated line scanning confocal fluorescentmicroscope (LSM) and analysed using a suite of novel image analysisalgorithms in order to identify miRNAs that were able to modulate HIVinfection. The present inventors consequently identified several miRNAmolecules that could be used in anti-HIV therapeutic approaches. Theinventors further identified specific functional pathways enriched inthe miRNA screen data. The functional pathways and miRNA targetsidentified by the initial screens therefore also represent potentiallyattractive points of intervention for targeted HIV therapies. Theinventors conducted targeted drug/compound screens focused on thepathways and protein targets identified in the miRNA screens. Using thisapproach the inventors were able to identify a number ofdrugs/compounds, which are able to either suppress HIV infection orpreferentially target or sensitise host cells to apoptose in response toinfection by HIV. A number of these drugs have also already beenapproved by the FDA for use in the treatment of various forms of cancerand this should facilitate the rapid repurposing of these compounds foruse as novel anti-HIV therapeutics.

The inventors have also described the use of a primary miRNA screen as atool to identify specific therapuetically relevant functional pathways(in this case pertinent to HIV replication). This approach allows forthe interogation of pharmaceutical compounds (preferbaly FDA-approved)targeted to these pathways, thus not only reducing the cost associatedwith large scale non-targeted compound screening but also dramaticallyincreasing the likehood of identifying compounds that will illicit thedesired biological effect.

As used herein the term “nucleic acid” refers to a deoxyribonucleotideor ribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses analogues of natural nucleotidesthat hybridise to nucleic acids in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence includes the complementary sequence thereof.

The present invention provides nucleic acid compounds which are usefulin the modulation of gene expression. The nucleic acid compounds of theinvention modulate gene expression by hybridising to nucleic acid targetsequences. The result of the hybridisation is the loss of normalfunction of the target nucleic acid. In a preferred embodiment of thisinvention modulation of gene expression is effected via modulation of aparticular RNA associated with the particular gene-derived RNA.

As used herein “RNA interference” (RNAi) is the process by whichsynthetic siRNAs or the expression of a nucleic acid (including miR,siRNA, shRNA) causes sequence-specific degradation of complementary RNA,sequence-specific translational suppression or transcriptional genesilencing and further as used herein “RNAi-encoding sequence” refers toa nucleic acid sequence which, when expressed, causes RNA interference.

The abbreviation “siRNA” refers to a “small interfering RNA”. siRNA'sgenerally consist of a short double-stranded RNA molecule, theantisense- (guide) strand and the sense- (passenger) strand. Typically asiRNA molecule comprises a duplex region with 3′ overhangs of 2nucleotides. One strand is incorporated into a cytoplasmic RNA-inducedsilencing complex (RISC). This directs the sequence specific RNAcleavage that is effected by RISC. Mismatches between the siRNA guideand its target may cause translational suppression instead of RNAcleavage. siRNA may be synthetic or derived from processing of aprecursor by Dicer.

Specifically the invention relates to the use of microRNAs (miRNAs). ThemiRNAs of the invention may be classified as miRNA mimics or miRNAinhibitors.

As used herein miRNA mimics refers to double-stranded, syntheticreplicates of endogenous miRNAs which augment the intracellularconcentration and function of a specific endogenous miRNA. miRNAinhibitors on the other hand refers to synthetic, single-stranded RNAmolecules which are able to bind to endogenous target miRNAs and preventthem from regulating their mRNA targets.

The term “transcription” refers to the process of producing RNA from aDNA template. “In vitro transcription” refers to the process oftranscription of a DNA sequence into RNA molecules using a laboratorymedium which contains an RNA polymerase and RNA precursors and“intracellular transcription” refers to the transcription of a DNAsequence into RNA molecules, within a living cell. Further, “in vivotranscription” refers to the process of transcription of a DNA sequenceinto RNA molecules, within a living organism.

As used herein, the term ‘target nucleic acid’ or “nucleic acid target”refers to a nucleic acid sequence derived from a gene, in respect ofwhich the RNAi encoding sequence of the invention is designed toinhibit, block or prevent gene expression, enzymatic activity orinteraction with other cellular or viral factors. In terms of theinvention “target nucleic acid” or “nucleic acid target” encompass anynucleic acid capable of being targeted including without limitationincluding DNA, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from DNA, and also cDNA derived from RNA.

The present invention provides nucleic acid compounds which are usefulin the modulation of gene expression. The nucleic acid compounds of theinvention modulate gene expression by hybridising to nucleic acid targetsequences. The result of the hybridisation is the loss of normalfunction of the target nucleic acid. In a preferred embodiment of thisinvention modulation of gene expression is effected via modulation of aparticular RNA associated with the particular gene-derived RNA. Thepresent invention further provides for pharmaceutical compounds whichtarget the particular pathways, polynucleotides and/or polypeptides in acell which are modulated by the nucleic acid compounds of the inventionincluding but not limited to inhibition of cellular processes,interference with cellular processes, interruption of biosyntheticpathways, apoptosis, interference with transcription, translation and/orreplication.

In the context of the present invention, “modulation” and “modulation ofexpression” can mean either an increase (stimulation) or a decrease(inhibition) in the level or amount of a nucleic acid molecule encodingthe gene, e.g., DNA or RNA. Inhibition is often the preferred form ofmodulation of expression and mRNA is often a preferred target nucleicacid.

The invention also relates to repurposing pharmaceutical compounds foruse in the treatment of HIV.

The term “pharmaceutically acceptable” refers to properties and/orsubstances which are acceptable for administration to a subject from apharmacological or toxicological point of view. Further“pharmaceutically acceptable” refers to factors such as formulation,stability, patient acceptance and bioavailability which will be known toa manufacturing pharmaceutical chemist from a physical/chemical point ofview.

The “suitable forms” of the pharmaceutical compounds may be combinedwith “pharmaceutically acceptable carriers” and other elements known inthe art in order to ensure efficient delivery of the activepharmaceutical ingredient to a subject.

By “pharmaceutically acceptable carrier” is meant a solid or liquidfiller, diluent or encapsulating substance which may be safely used forthe administration of the extract, pharmaceutical composition and/ormedicament to a subject.

The term “effective amount” in the context of preventing or treating acondition refers to the administration of an amount of the activepharmaceutical ingredient in a pharmaceutical compound to an individualin need of treatment, either a single dose or several doses of thepharmaceutical compound may be administered to a subject.

Although some indications have been given as to suitable dosages of thepharmaceutical compounds of the invention, the exact dosage andfrequency of administration of the effective amount will be dependent onseveral factors. These factors include the individual components used,the formulation of the extract or pharmaceutical composition containingthe extract, the condition being treated, the severity of the condition,the age, weight, health and general physical condition of the subjectbeing treated, and other medication that the subject may be taking, andother factors as are known to those skilled in the art. It is expectedthat the effective amount will fall within a relatively broad range thatcan be determined through routine trials.

Toxicity and therapeutic efficacy of compositions of the invention maybe determined by standard pharmaceutical procedures in cell culture orusing experimental animals, such as by determining the LD50 and theED50. Data obtained from the cell cultures and/or animal studies may beused to formulate a dosage range for use in a subject. The dosage of anycomposition of the invention lies preferably within a range ofcirculating concentrations that include the ED50 but which has little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilised. Forcompositions of the present invention, the therapeutically effectivedose may be estimated initially from cell culture assays.

The following example is offered by way of illustration and not by wayof limitation.

EXAMPLE

Cell Lines

The miRNA and targeted-drug/compound screens were performed using theengineered GHOST(3) cell line. The GHOST(3) cell line was originallyderived from a Human Osteosarcoma cell line (HOS) and is available fromthe NIH AIDS Research and Reference Reagent Program (catalogue number.3942). GHOST(3) cells were seeded at a density of ˜6000 cells per wellin uClear 96 well imaging plates (Grenier Bio-one) for all screeningapplications. HOS cells were stably transduced with an MV7neo-T4retroviral vector and stably express an HIV-2 Long terminal Repeat (LTR)promoter driving and hGFP and a cytomegalovirus (CMV) promoter drivingHygromycin B resistance (Momer et al., (1999)). Typical HIV infection inGHOST(3) cells results in the binding of the HIV trans-activatingprotein (tat) to the LTR promoter thus stimulating hGFP expression ininfected cells. This reporter cell line has previously been shown to besensitive to multiple HIV-1 subtypes in addition to HIV-2 and has alsobeen validated as tool to directly assay HIV-1 infection in culturedcells (Cecilia et al., (1998)). GHOST(3) cells were cultured inDulbecco's Minimal Essential Medium (DMEM, Invitrogen) supplemented with10% heat-inactivated Fetal bovine serum (FBS) (Biochrom) and 1×GlutaMax, stabilized L-glutamine reagent (Invitrogen). GHOST(3) cellswere cultured under antibiotic selection with Puromycin (1 ug/mL);Hygromycin B (100 ug/mL) and G418 (500 ug/mL). The human embryonicKidney 293T (HEK293T) and JC53 cell lines were cultured in DMEMsupplemented with 10% heat-inactivated Fetal bovine serum (FBS)(Biochrom) and 1× GlutaMax (Invitrogen).

miRNA Mimic and Inhibitors

The miRNA screens were conducted using the miRIDIAN® miRNA mimic andinhibitor libraries (Thermo Scientific™) based on miRBase v.16.0(www.miRBase.org). The library was comprised of 1239 miRNA mimicmolecules and 1245 miRNA inhibitor molecules. miRNA mimics aredouble-stranded, synthetic replicates of endogenous miRNAs and augmentthe intracellular concentration and function of a specific endogenousmiRNA (FIG. 1). miRNA inhibitors are synthetic, single-stranded RNAmolecules which are able to bind to endogenous target miRNAs and thusprevent them from regulating their mRNA targets (FIG. 1). miRNA mimicand inhibitor molecules were used at a final concentration of 25 nM andintroduced into cell lines by liposome-mediated transfection usingLipofectamine RNAiMax (Invitrogen). Briefly. 2.5 uL of 1 uM miRNA mimicor inhibitor molecules were diluted to final volume of 10 uL inserum-free media (DMEM, Gibco). A volume of 0.15 uL of RNAiMax was thendiluted in serum free media to a final volume of 10 uL. The 10 uL ofdiluted RNAiMax solution was then combined with the diluted miRNAsolution and this transfection mixture was incubated at room temperaturefor 20 minutes. The 20 uL transfection mixture was then added directlyGHOST(3) cells cultured in a well of a 96 well. Suitable control wellswere included on each plate and these were used in the subsequent imageanalysis steps.

Targeted Drug Screens

A library of 101 Food and Drug Administration (FDA) approved oncologydrugs as well as a library of 192 kinase-inhibitor compounds werescreened as potential anti-HIV therapeutics using the GHOST(3) reportercell line. Drugs were screened at 3 different concentrations of 100 nM,1 uM and 10 uM. Each library was screened in triplicate at each drugconcentration and in the following 3 formats:

i) Phenocopy Screen

This screen format was utilised to identify drugs/compounds that wereable to protect host cells against HIV infection; suppress HIVreplication or selectively sensitize or prime host cells to apoptoseupon infection by HIV. GHOST(3) cells were seeded in the wells of a 96well plate 24 hour prior to addition of a specific oncology drug at theappropriate concentration. Twenty-four hours post drug treatment, thedrug supplemented media was removed and replaced with infective mediacontaining the Subtype B pseudoviral strain, PSGIII. Cells were infectedat an MOI of 0.5. The infective media was removed 48 hour post infectionand cells were processed for imaging as previously described (See SamplePreparation).

ii) Clearance Screen

This screen format was utilised to identify drugs/compounds that wereable to suppress HIV replication in host cells already infected by HIVor drugs/compounds that were able to selectively target HIV-infectedcells for apoptosis. GHOST(3) cells were seeded in the wells of a 96well plate 24 hour prior to infection with the Subtype B pseudoviralstrain, PSGIII, at an MOI of 0.5. The infective media was then removed48 hour post infection and replaced with normal culture mediasupplemented with a drug/compound at the appropriate concentration.Twenty-four hours post drug treatment, cells were processed for imagingas previously described.

iii) Drug Only Screen

This screen format was used as a control to identify drugs that werebroadly cytotoxic. Drugs/compounds that resulted in >25% cytotoxicitycompared to untreated control wells were eliminated from furtheranalysis. GHOST(3) cells were seeded in 96 well plates, 24 hr postseeding, drugs were added to culture medium at the required finalconcentration. Twenty-four hours post drug treatment cells wereprocessed for imaging as described previously.

Viral Production and Infection Assay

Subtype B viral stocks were produced by the dual transfection of HEK293Tcells with a PSGIII (Subtype B) viral backbone plasmid which lacks anenvelope/packaging protein and a second plasmid expressing an HIV-BALviral envelope protein (Subtype B). Plasmid transfections were conductedusing the FuGene 6 lipid-based transfection reagent (Promega) as per themanufacturer's guidelines. Viral titres were determined using aluciferase readout-assay based on the JC53 cell line and the TCID50calculation method described by Reed and Muench (1938). GHOST(3) cellswere exposed to virions at an MOI of 0.5 for both the miRNA and targeteddrug/compound screens. Cells were infected in SFM supplemented with 1×Glutamax (Infecting media) as FBS has previously been described tonegatively impact the infection efficiency of HIV.

Sample Preparation

Cells were Washed Twice in 1×PBS Before Fixation with a 3.7%Formaldehyde

solution for 30 minutes at room temperature. Cells were washed once with1×PBS before the addition of Hoechst Nuclear stain (Invitrogen) at 2 uMfinal concentration in 1×PBS for 30 minutes at room temperature. TheHoechst staining solution was aspirated and replaced with 50 uL of 1×PBSsolution before imaging.

Imaging

Four fields of view (100 um apart) were acquired from the central regionof each well imaged. Images were acquired in the 405 and 488 fluorescentchannels for each field of view. Wells were imaged using the 20× ewldobjective on the Image Xpress Ultra automated confocal microscope system(Molecular Devices). The imaging parameters were kept constant for allreplicates of both miRNA and targeted drug/compound screens.

Image Analysis

HCS analyses rely on the discrimination of multiple biologicaldescriptors between images derived from experimental and control wellsfor HIT identification. HCS analyses thus require the extractionquantitative, subcellular measurements (features) from the acquiredimages in order to identify subsets of these features that canaccurately and reproducibly classify specific biological phenotypeswithin experimental wells.

Feature extraction algorithms were utilized in order to extract 16subcellular measurements from the acquired images. These 16 factors arederived from 15 descriptors previously described by Genovesio et al.2011 to be relevant indicators of the infective state of host cells,with addition of ‘infection efficiency’ as a descriptor of thepercentage of infected cells per treatment well.

A bespoke support vector machine-learning (SVML) pattern recognitionalgorithm was trained to identify phenotypes of interest using therelevant control wells. Transfection of GHOST(3) with an siRNA targetingthe human CD4 receptor (25 nM) 48 hours prior to exposure to HIV wasshown to significantly suppress HIV infection and produce a visuallydistinct phenotype representative of the inhibition of HIV replication.Knockdown of the CD4 receptor required for HIV entry in GHOST(3) cells48 hours prior to exposure to HIV resulted in suppressed GFP reportersignal and when compared to natural infection controls (high GFP signal)creates contrasting visual phenotypes required for subsequent imageanalysis. siCD4 wells were included on every screening plate in themiRNA screens as positive controls for suppressed HIV infection.Likewise the addition of the HIV-1 integrase inhibitor, Raltegravir (10uM) to GHOST(3) culture medium 24 hour prior to exposure to HIV was alsoshown to produce a distinct phenotype representative of suppressed HIVreplication. The transfection of siCD4 and Raltegravir treatment werethus utilised in the miRNA and drug/compound screens respectively aspositive controls for the inhibition of HIV-1 replication.

SVML algorithms were trained using these controls and were thus able togenerate a decision boundary (HIT boundary) that defines a region of 16dimensional space which is representative of the Raltegravir or siCD4control wells and which is also distinct from the controlsrepresentative of a natural infection phenotype. Experimental wells fromthe miRNA and drug screens, which were classified within the HITboundary closer to the siCD4 and Raltegravir controls, were identifiedas positive HITs for the inhibition of HIV replication. miRNA mimics orinhibitors which were able to enhance activation of the HIV LTR promoterwere identified by evaluating the average GFP intensity in experimentalwells, relative to the natural infection controls. miRNA molecules whichproduced a greater than 2-fold increase in average GFP intensityrelative to control wells were classified as HIV enhancer miRNAmolecules. Only miRNA molecules and drugs which were shown to elicitconcordant phenotypes across replicate screens were included in thefinal list of HITs. Additionally, using the values obtained for cellcounts per well (as per Hoechst staining) we were also able to evaluatethe cytoxicity of specific drugs and compounds using the “drug only”screen format as a control. Drugs/compounds which were shown to becytotoxic specifically in response to HIV exposure in either thephenocopy and clearance screen formats but not in the drug only screenswere also identified as drugs/compounds of interest.

Host miRNAs are Able to Regulate HIV Replication and Modulate Host CellResponses to HIV Infection

The use of both miRNA mimic and inhibitor molecules allowed for theparallel evaluation of both over-expression and suppression phenotypesfor a given miRNA. This two-pronged functional approach allows for arobust assessment of the functional relevance of a given miRNA incontext of HIV infection. The bespoke image analyses identified a numberof miRNA mimic and inhibitor molecules which could potentially modulateHIV infection (See Table 1). These miRNA molecules could broadly bedivided into three classes depending on their effect on host cells inresponse to exposure to HIV:

-   -   I. miRNA molecules which are able to inhibit HIV entry or prime        uninfected cells to suppress LTR activation upon infection by        HIV    -   II. miRNA molecules which are able to sensitize uninfected cells        to apoptosis in response to infection by HIV    -   III. miRNA molecules which could promote activation of the HIV        LTR promoter.

TABLE 1Human microRNA mimics and inhibitors that reguate HIV replicationand infection miRNA molecule miRNA miRNA name Molecule sequenceEffect^(a) Target(s) Pathway^(*) hsa-let-7a-5p Mimic UGA GGU AGU AGGSupp CDK6, EGFR, DDR, CC, UUG UAU AGU U CDKN1A APO, PC (SEQ ID NO: 1)hsa-let-7d-5p Mimic AGA GGU AGU AGG Supp HMGA2, H2AFX DDR, PCUUG CAU AGU U (SEQ ID NO: 2) hsa-miR-23a Mimic AUC ACA UUG CCA SuppFANCG DDR GGG AUU UCC (SEQ ID NO: 3) hsa-mir-29c* Mimic UGA CCG AUU UCUTox BCL-xL APO, PC CCU GGU GUU C (SEQ ID NO: 4) hsa-miR-34c-3p MimicAAU CAC UAA CCA EH MET, CDK4, CC, APO, PC CAC GGC CAG G BCL.2(SEQ ID NO: 5) hsa-miR-92a-1-5p Mimic AGG UUG GGA UCG Supp Unknown PCGUU GCA AUG CU (SEQ ID NO: 6) hsa-miR-124a-3p Mimic UAA GGC ACG CGG SuppNGR1, CDK6 DDR, PC UGA AUG CC (SEQ ID NO: 7) hsa-miR-125b-5p MimicUCC CUG AGA CCC EH P53 DDR, CC UAA CUU GUG A APO, PC (SEQ ID NO: 8)hsa-miR-138 Mimic GC UGG UGU UGU Supp H2AX DDR GAA UCA GGC CG(SEQ ID NO: 9) hsa-miR-146a Mimic UGA GAA CUG AAU Supp BRCA1,DDR, CC, PC UCC AUG GGU U BRCA2, (SEQ ID NO: 10) ERBB4, CDKN1Ahsa-miR-149-3p Mimic AGG GAG GGA CGG Supp AKT1, EF2F1 CC, PCGGG CUG UGC (SEQ ID NO: 11) hsa-miR-150 Inhibitor UCU CCC AAC CCU SuppP53 DDR, APO, UGU ACC AGU G PC (SEQ ID NO: 12) hsa-miR-150 MimicUCU CCC AAC CCU EH P53 DDR, APO, UGU ACC AGU G PC (SEQ ID NO: 13)hsa-miR-155 Mimic UUA AUG CUA AUC EH P53 PC, APO GUG AUA GGG GU(SEQ ID NO: 14) hsa-miR-193b-5p Mimic CGG GGU UUU GAG Supp, RB1, DHFRDDR, CC, PC GGC GAG AUG A Tox (SEQ ID NO: 15) hsa-miR-200c MimicUAA UAC UGC CGG EH BCL-2, XIAP DDR, APO GUA AUG AUG GA (SEQ ID NO: 16)hsa-miR-342-5p Mimic AGG GGU GCU AUC Supp Unknown PC UGU GAU UGA (SEQID NO: 17) hsa-miR-381-5p Mimic UUA UCA GAPS UCU EH Unknown PCCCA GGG GUA C (SEQ ID NO: 18) hsa-miR-421 Mimic AUC AAC AGA CAU Supp,ATM DDR UAA UUG GGC GC Tox (SEQ ID NO: 19) hsa-miR-423-3p MimicAGC UCG GUC UGA Supp CDKN1A DDR, CC, PC GGC CCC UCA GU (SEQ ID NO: 20)hsa-miR-504 Mimic AGA CCC UGG UCU EH P53, BAX, FAS DDR, CC,GCA CUC UAU C APO PC (SEQ ID NO: 21) hsa-miR-509-3p MimicUGA UUG GUA CGU Supp Unknown PC CUG UGG GUA G (SEQ ID NO: 22)hsa-miR-637 Mimic ACU GGG GGC UUU Supp SP7, STAT3, DDR, CCCGG GCU CUG CGU LIF (SEQ ID NO: 23) hsa-mir-650 Mimic AGG AGG CAG CCCSupp NG4, NDRG2 DDR, CC, PC UCU CAG GAC (SEQ ID NO: 24) hsa-miR-520d-5pMimic CUA CAA AGG GAA Supp Unknown PC GCC CUU UC (SEQ ID NO: 25)hsa-miR-1200 Mimic CUC CUG AGC CAU EH NEIL2 PC UCU GAG CCU C(SEQ ID NO: 26) hsa-miR-1908 Mimic CGG CGG GGA CGG Supp, Unknown PCCGA UUG GUC (SEQ Tox ID NO: 27) hsa-miR-1910 Mimic CCA GUC CUG UGC EHUknown PC CUG CCG CCU (SEQ ID NO: 28) hsa-miR-2110 Mimic UUG GGG AAA CGGSupp Unknown PC CCG CUG AGU G (SEQ ID NO: 29) hsa-miR-3162 MimicUUA GGG AU AGA Supp Unknown PC AGG GUG GGG AG (SEQ ID NO: 30)hsa-miR-3185 Mimic AGA AGA AGG CGG Supp Uknown PC UCG GUC UGC GG(SEQ ID NO: 31) hsa-miR-3189 Mimic CCC UUG GGU CUG Supp, Uknown PCAUG GGG UAG (SEQ Tox ID NO: 32) hsa-miR-3191 Mimic UGG GGA CGU AGC SuppUnknown PC UGG CCA GAC AG (SEQ ID NO: 33) hsa-miR-4259 MimicCAG UUG GGU CUA Supp Unknown PC GGG GUC AGG A (SEQ ID NO: 34)hsa-miR-4314 Mimic CUC UGG GAA AUG Supp Unknown PC GGA CAG (SEQ IDNO: 35) ^(a)Supp = suppressed HIV replication; EH = enhanced HVreplication; Tox = Toxic in response to HIV infection ^(*)DDR = DNAdamage response; CC = cell cycle; APO = apoptosis; PC = pathways incancer

Visual inspection of the experimental wells classified as HITs by ouranalysis confirmed that these wells were correctly classified by theanalysis algorithms. miRNAs identified as inhibitory or protectiveagainst HIV were shown to be to visually similar to the siCD4 controlsyielding reduced GFP reporter signal, whereas HITs identified asenhancers were shown to exhibit much stronger activation of the HIV LTRas equated to GFP signal intensity yielding significantly higher GFPreporter signal than natural infection control wells. This served as avalidation of the image analysis algorithms abilities to accuratelyclassify experimental wells relative to the HIV infective state ofGHOST(3) cells.

We have uncovered a number of previously unpublished miRNA-HIVinteractions and have also identified a number of miRNA-based moleculeswith potent anti-HIV activity whose endogenous targets and functionshave not yet been elucidated. Further analysis of the miRNA HITsrevealed an enrichment of miRNAs associated with a very specific subsetof functional pathways. Using a combination of bioinformatics basedmanual searches and online databases of experimentally validated miRNApathway and miRNA-target associations, such as miRpath (S. Vlachos et al((2012)) and miRTarBase v.4.5 (Hsu (2011)), we were able to identify theDNA damage, cell cycle and apoptotic pathways as critical points ofintervention in the HIV replicative cycle with the majority of miRNAsand miRNA targets identified in the screen shown to be functionallyactive in these pathways.

Characterisation of Oncogenic Traits Associated with HIV Infection

A well-characterised feature of the HIV replicative cycle involves theintegration of the HIV proviral genome into host chromatin. In order toachieve this, the virus must induce a double strand break (DSB) in thehost DNA and recruit host DNA repair machinery to the break sites,resulting in integration of the provirus into the host genome. DSBs arethe most detrimental form of DNA lesion and as there is no intactcomplementary strand to serve as a template for repair, DSBs are poorlytolerated (Khanna, 2001). A single DSB is sufficient to kill yeast cellsif it inactivates an essential gene, or trigger apoptosis in metazoancells. If left uncorrected, DSBs can result in chromosomal breakage ortranslocations that lead to developmental defects, neurodegeneration,immunodeficiency and cancer. As the infecting virus cannot coordinatethe number of integration events and consequent DSBs per host cell, HIVmust have evolved a mechanism to either mask the DSB from the cell orcarefully orchestrate any cellular response to the DSB to avoidtriggering apoptosis. Furthermore, to induce a DSB yet ensure survival,HIV must take control of prosurvival mechanisms and suppress activationof pro-apoptotic genes.

In order to evaluate the relationship between HIV-mediated DSBs and thecontrol of cellular DNA damage responses, we conducted 2 sets ofexperiments. In the first experiment, cells were treated with known DNAdamage-inducing agents prior to HIV infection. Similar to cells treatedonly with DNA damage-inducing agents, these cells underwent completeapoptosis due to drug-induced cytotoxicity. In contrast, cells that wereinfected with HIV 24 hours prior to the addition of the DNAdamage-inducing agents were seemingly able to evade apoptosis in spiteof high levels of DNA damage. In addition, these cells stained positivefor p53 serine 46, which is specifically activated in cells directedtowards apoptosis, suggesting that HIV is able to evade the cellularapoptotic response by manipulating the pathway downstream of p53. Thesefindings support the results of our miRNA screens, which suggested thatHIV requires and maintains tight regulation over the DNA damage repair,cell-cycle and apoptotic pathways. Our DNA damage-inducing experimentaldata also suggests that HIV actively manipulates these pathways toproduce a pro-survival phenotype in cells that would naturally undergoapoptosis. This cancer-like characteristic of HIV infection would thussuggest that drugs targeting pathways associated with cancers may alsoprove effective against HIV.

Targeted Drug Screens for the Repurposing of Existing Drugs/Compoundsfor the Treatment of HIV

The identification numerous miRNAs and miRNA targets linked to DNADamage Repair, cell cycle and apoptotic pathways in addition to observedcancerous nature of HIV infection in GHOST(3) suggests a closesimilarity between HIV-1 infection and many models of cancer. We soughtto exploit this cancer-like nature of HIV via the use of a targetedscreening approach based on a library of FDA-approved anticancer drugsand a library of Kinase Inhibitors. We screened these libraries usingthe GHOST(3) and HC screening approach that was used for the initialmiRNA screens. A number of FDA approved anti-cancer compounds (Table 2.)as well a number of kinase inhibitor molecules (Table 3.) wereidentified as putative HITs by image analyses. Visual inspection ofthese HITs again confirmed that our analysis approach was able tocorrectly identify and classify the wells of interest. We separated thephenotypes/effects of the drugs HITs into 4 functional classes:

-   -   I. Drugs which are selectively toxic to host cells already        infected by HIV    -   II. Drugs which are able to sensitize uninfected cells to        apoptosis in response to infection by HIV    -   III. Drugs which are able to suppress HIV LTR activity in host        cells already infected with HIV    -   IV. Drugs which are able to inhibit HIV entry or prime        uninfected cells to suppress LTR activation upon infection by        HIV

TABLE 2 Pharmaceutical compounds of interest Approved Drug Name useEffect* Target or Mechanism of Action A-674563 Kinase Suppressed HIV^(b)A-674563 is a potent selective protein inhibitor & Toxic in kinase B/Aktinhibitor with an IC50 of response to HIV^(a) 14 nM. AbirateroneOncology Toxic in response Androgen biosynthesis inhibitor, which toHIV^(a) inhibits 17α hydroxylase Aminolevulinic Oncology Toxic inresponse A metabolic precursor of acid to HIV^(a) protoporphyrin IX(PpIX), which is a hydrochloride photosensitizer AT7519 Kinase Toxic inresponse CDK1/cyclinB, CDK2/CyclinA, inhibitor to HIV^(a) CDK2/Cyclin E,CDk4/CyclinD1, CDK6/Cyclin D3, CDk5/p35. Aurora A Kinase Toxic inresponse (Aurora A: IC50 at 0.0034 μM; Aurora Inhibitor I inhibitor toHIV^(a) B: IC50 at 3.4 μM), (B/A ratio = 1000). BIBF1120 KinaseSuppressed HIV^(a) (VEGFR), PDGFR and FGFR kinase (Vargatef) inhibitorinhibitor to VEGFR1, VEGFR2, VEGFR3 with IC50 of 34, 13 and 13 nM,respectively. Cisplatin Oncology Suppressed HIV^(b) Platinum-containingdrug that reacts in vivo by binding to and causing crosslinking of DNA,which ultimately triggers apoptosis Crenolanib (CP- Kinase Toxic inresponse Potent PDGFR-α inhibitor with IC50 of 868596) inhibitor toHIV^(a) 0.9 and 1.8 nM against PDGFRα and PDGFRβ, respectively.Dactinomycin Oncology Suppressed HIV^(b) Good evidence exists that thisdrug bind strongly, but reversibly, to DNA, interfering with synthesisof RNA (prevention of RNA polymerase elongation) and, consequently, withprotein synthesis. Dexrazoxane Oncology Suppressed HIV^(b) Ring-openedbidentate chelating agent that chelates to free iron and interferes withiron-mediated free radical generation Erlotinib Oncology Toxic inresponse The mechanism of clinical antitumor hydrochloride to HIV^(a)action of erlotinib is not fully characterized. Erlotinib inhibits theintracellular phosphorylation of tyrosine kinase associated with theepidermal growth factor receptor (EGFR) Everolimus Oncology SuppressedHIV^(b) Inhibitor of mammalian target of rapamycin (MTOR) FloxuridineOncology Toxic in response The primary effect is interference with toHIV^(a) DNA synthesis and to a lesser extent, inhibition of RNAformation through the drug's incorporation into RNA, thus leading to theproduction of fraudulent RNA. Fluorouracil also inhibits uracil ribosidephophorylase, which prevents the utilization of preformed uracil in RNAsynthesis Foretinib Kinase Suppressed HIV^(a) MET and VEGFR2/KDR kinasesinhibitor inhibitor with an IC50 of 0.4 and 0.8 nM for MET and KDR,respectively. Gefitinib Oncology Toxic in response Inhibits theepidermal growth factor to HIV^(a) receptor (EGFR) tyrosine kinase bybinding to the adenosine triphosphate (ATP)-binding site of the enzymeGSK2126458 Kinase Suppressed HIV^(a) Highly potent PI3K and mTORinhibitor inhibitor with an app Ki of 19 pM for PI3K. IfosfamideOncology Suppressed HIV^(b) The cytotoxic action is primarily throughthe alkylation of DNA, done by attaching the N-7 position of guanine toits reactive electrophilic groups. The formation of inter- and intra-strand cross-links in the DNA results in cell death. LDN193189 KinaseToxic in response BMP inhibitor with IC50 of 5 and 30 nM inhibitor toHIV^(a) for ALK2 and ALK3, respectively. NVP-BHG712 Kinase SuppressedHIV^(a) EphB4, VEGFR2, c-raf, c-src and c-Abl inhibitor kinase inhibitorwith ED50 of 25 nM, 4.2, 0.4, 1.3 and 1.7 μM, respectively. PIK-75Kinase Toxic in response p110α/γ forms of PI3K inhibitor with inhibitorto HIV^(a) IC50 of 6, 1300, 76, 510 nM for p110α, p110β, p110γ, p110δ,respectively. Plicamycin Oncology Toxic in response Antineoplasticantibiotic produced by to HIV^(a) Streptomyces plicatus, Plicamycin ispresumed to inhibit cellular and enzymic RNA synthesis by forming acomplex with DNA. Plicamycin may also lower calcium serum levels byinhibiting the effect of parathyroid hormone upon osteoclasts or byblocking the hypercalcemic action of pharmacologic doses of vitamin DPonatinib Kinase Suppressed HIV^(a) pan-BCR-ABL, mutated form, inhibitorVEGFR2, FGFR1, PDGFRα, mutant FLT3 phosphorylation and LYN. SaracatinibKinase Suppressed HIV^(a) Dual-specific Src/Abl kinase inhibitorinhibitor with IC50 of 2.7 and 30 nM for c-Src and Abl kinase,respectively. SNS-032 (BMS- Kinase Toxic in response (CDK) 9, 7 and 2inhibitor with IC50 of 387032) inhibitor to HIV^(a) 4, 62 and 38 nM forCDK9, CDK2/cyclin A and CDK7/Cyclin H. Temozolomide Oncology SuppressedHIV^(a) Undergoes spontaneous intracellular conversion via hydrolysisinto a potent methylating agent, MTIC.4 MTIC methylates a number ofnucleobases, most important, the guanine base. This results in theformation of nicks in the DNA, followed by apoptosis, because cellularrepair mechanisms are unable to adjust to the methylated baseThalidomide Oncology Suppressed HIV^(a) Thalidomide can directly inhibitangiogenesis induced by beg or VEGF, Derivative Pomalidomide approved byFDA in 2013 Vemurafenib Oncology Suppressed HIV^(b) Inhibitor of theactivity of some forms of the mutant BRAF protein, including BRAF V600EVincristine Oncology Suppressed HIV^(a) Inhibitor of mitosis atmetaphase sulfate through its interaction with tubulin. Like other vincaalkaloids, Vincristine may also interfere with: 1) amino acid, cyclicAMP, and glutathione metabolism, 2) calmodulin-dependent Ca2+-transportATPase activity, 3) cellular respiration, and 4) nucleic acid and lipidbiosynthesis. Vorinostat Oncology Toxic in response Inhibits theenzymatic activity of to HIV^(a) histone deacetylases HDAC1, HDAC2 andHDAC3 (Class I) and HDAC6 (Class II) at nanomolar concentrations (IC50 <86 nM). WZ4002 Kinase Suppressed HIV^(a) EGFR T790M inhibitor(IC50 < 20nM). inhibitor *Key to Drug Effect Phenotypes: Suppressed HIV^(a) = inPhenocopy Screen (Protects against infection, suppresses new infections)Suppressed HIV^(b) = in Clearance Screen (Suppresses establishedinfection) Toxic in response to HIV^(a) = in Phenocopy Screen(Sensitization of host cells to apoptosis in response to HIV infection)

GHOST(3) cells treated with the drugs/compounds of interest surprisinglyrevealed reduced levels of GFP-reporter signal in both stably infectedHIV cells as well naïve cells treated prior to exposure to HIV. Brieflya number of drugs/compounds were identified which were able to suppressHIV replication as well target HIV infected cells for apoptosis.

The pathways and targets of the HITs identified by the drug/compoundscreens also showed a strong overlap with the pathways and targetsidentified in the miRNA screens. These findings again support the notionthat specific signalling cascades or nodes within the DNA damageresponse, cell cycle and apoptotic pathways may prove highly effectiveas targets for host-directed anti-HIV ART intervention strategies.

REFERENCES

-   Azuma-Mukai, A., et al. Proc Natl Acad Sci USA, 2008. 105(23): p.    7964-9.-   Baek, D., et al. Nature, 2008. 455(7209): p. 64-71.-   Barbato, C., et al. J Biomed Biotechnol, 2009. 2009: p. 803069.-   Cecilia, D., et al. Journal of Virology. 1998 72(9): 6988-6996.-   Cullen, B. R. Nat Immunol, 2013. 14(3): p. 205-10.-   Farh, K. K., et al. Science, 2005. 310(5755): p. 1817-21.-   Ebert, M. S. and P. A. Sharp. Cell, 2012. 149(3): p. 515-24.-   Fire, A. et al. Nature, 1998. 391(6669): p. 806-11.-   Genovesio, A. Journal of Biomolecular Screening. 2011 16(9):    945-958.-   Hsu, S.-D. Nucleic Acids Research, 2011 39 (SUPPL. 1), pp. D163-D169-   Lander, E. S., et al. Nature, 2001. 409(6822): p. 860-921.-   Lee, Y., et al. EMBO J, 2004. 23(20): p. 4051-60.-   Lee, Y., et al. EMBO J, 2002. 21(17): p. 4663-70.-   Lim, L. P., et al. Nature, 2005. 433(7027): p. 769-73.-   Liu, J., et al. Science, 2004. 305(5689): p. 1437-41.-   Martinez, J. and T. Tuschl. Genes Dev, 2004. 18(9): p. 975-80.-   Morner, A., et al. Journal of Virology. 1999 73(3): 2343-2349.-   Ponting, C. P. and T. G. Belgard. Hum Mol Genet, 2010. 19(R2): p.    R162-8.-   Reed, L. J. and Muench, H. The American Journal of Hygiene. 1938:    27: p 493-497.-   S. Vlachos, N. et al. Nucleic Acids Research 2012. 40: W498-504.-   Yamada, K. et al. Science, 2003. 302(5646): p. 842-6.-   Yekta, S. Science, 2004. 304(5670): p. 594-6.

The invention claimed is:
 1. A method of inhibiting HIV entry or priminguninfected cells to suppress LTR activation upon infection by HIV in asubject comprising administering an anti-cancer compound or apharmaceutically acceptable salt thereof to the subject, wherein theanti-cancer compound or pharmaceutically effective salt thereof isforetinib,2,4-difluoro-N-[2-methoxy-5-(4-pyridazin-4-ylquinolin-6-yl)pyridin-3-yl]benzenesulfonamide,4-methyl-3-[(1-methyl-6-pyridin-3-ylpyrazolo[3,4-d]pyrimidin-4-yl)amino]-N-[3-(trifluoromethyl)phenyl]benzamide,ponatinib, saracatinib, vargatef, and/orN-(3-(5-chloro-2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide.2. A method of treating HIV infection in a subject comprisingadministering an anti-cancer compound or a pharmaceutically acceptablesalt thereof to the subject, wherein the anti-cancer compound orpharmaceutically effective salt thereof suppresses HIV LTR activityand/or induces apoptosis in a cell in response to HIV infection of thecell, wherein the anti-cancer compound or pharmaceutically acceptablesalt thereof that suppresses HIV LTR activity is(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine,dexrazoxane, everolimus, and/or vemurafenib, and wherein the anti-cancercompound or pharmaceutically acceptable salt thereof that inducesapoptosis in a cell in response to HIV infection of the cell is(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine,abiraterone,4-[(2,6-dichlorobenzoyl)amino]-N-piperidin-4-yl-1H-pyrazole-5-carboxamide,N-(2-Chlorophenyl)-4-(2-(4-(2-(4-ethylpiperazin-1-yl)-2-oxoethyl)phenylamino)-5-fluoropyrimidin-4-ylamino)benzamide,crenolanib, gefitinib,4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline,N-[(6-bromo-3-imidazo[1,2-a]pyridinyl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide,plicamycin, and/orN-[5-[(5-tert-butyl-1,3-oxazol-2-yl)methylsulfanyl]-1,3-thiazol-2-yl]piperidine-4-carboxamide.3. The method of claim 1, wherein the anti-cancer compound is foretinib.4. The method of claim 1, wherein the anti-cancer compound is2,4-difluoro-N-[2-methoxy-5-(4-pyridazin-4-ylquinolin-6-yl)pyridin-3-yl]benzenesulfonamide.5. The method of claim 1, wherein the anti-cancer compound is4-methyl-3-[(1-methyl-6-pyridin-3-ylpyrazolo[3,4-d]pyrimidin-4-yl)amino]-N-[3-(trifluoromethyl)phenyl]benzamide.6. The method of claim 1, wherein the anti-cancer compound is ponatinib.7. The method of claim 1, wherein the anti-cancer compound issaracatinib.
 8. The method of claim 1, wherein the anti-cancer compoundisN-(3-(5-chloro-2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide.9. The method of claim 2, wherein the anti-cancer compound thatsuppresses HIV LTR activity is(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine.10. The method of claim 2, wherein the anti-cancer compound suppressesHIV LTR activity and is dexrazoxane.
 11. The method of claim 2, whereinthe anti-cancer compound suppresses HIV LTR activity and is everolimus.12. The method of claim 2, wherein the anti-cancer compound suppressesHIV LTR activity and is vemurafenib.
 13. The method of claim 2, whereinthe anti-cancer compound induces apoptosis in the cell and is(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine.14. The method of claim 2, wherein the anti-cancer compound inducesapoptosis in the cell and is abiraterone.
 15. The method of claim 2,wherein the anti-cancer compound induces apoptosis in the cell and is4-[(2,6-dichlorobenzoyl)amino]-N-piperidin-4-yl-1H-pyrazole-5-carboxamide.16. The method of claim 2, wherein the anti-cancer compound inducesapoptosis in the cell and isN-(2-chlorophenyl)-4-(2-(4-(2-(4-ethylpiperazin-1-yl)-2-oxoethyl)phenylamino)-5-fluoropyrimidin-4-ylamino)benzamide.17. The method of claim 2, wherein the anti-cancer compound inducesapoptosis in the cell and is crenolanib.
 18. The method of claim 2,wherein the anti-cancer compound induces apoptosis in the cell and isgefitinib.
 19. The method of claim 2, wherein the anti-cancer compoundthat induces apoptosis in the cell is4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline.20. The method of claim 2, wherein the anti-cancer compound inducesapoptosis in the cell and isN-[(6-bromo-3-imidazo[1,2-a]pyridinyl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide.21. The method of claim 2, wherein the anti-cancer compound inducesapoptosis in the cell and is plicamycin.
 22. The method of claim 2,wherein the anti-cancer compound induces apoptosis in the cell and isN-[5-[(5-tert-butyl-1,3-oxazol-2-yl)methylsulfanyl]-13-thiazol-2-yl]piperidine-4-carboxamide.